Capillary zone electrophoresis ("CZE") in small capillaries (less than or equal to 75 .mu.) was first demonstrated by Jorgenson and Lukacs, and has proven useful as an efficient method for the separation of small solutes. J. Chromatog., 218 (1981), page 209; Anal. Chem., 53 (1981), page 1298. The mass transport of charged species past a single point of detection relies upon an electroosmosis effect generally described as the flow of a liquid in contact with a solid surface under the influence of a tangentially applied electric field. Attractive factors for electrophoretic separations by capillary zone electrophoresis are the small sample sizes, little or no sample pretreatment, and the potential for quantification and recovery of biologically active samples. For example, U.S. Pat. No. 4,675,300, inventors Zare et al., issued Jun. 23, 1987, describes theories and equipment for electrokinetic separation processes employing a laser-excited fluorescence detector. The system described by Zare et al. includes fused silica capillary with a 75 .mu. inside diameter.
High performance capillary electrophoresis ("HPCE") afforded the potential of extraordinary high efficiency separations of macromolecules. Unfortunately, the high efficiency first anticipated was based on the assumption that there would be no macromolecular/column interaction term in the unpacked (open tubular) column technique. It has been suggested by theoretical studies, which have been supported by experimental evaluation, Swedberg et al., (1989) in Techniques in Protein Chemistry (Hugli, ed.) Academic Press, San Diego, that this interaction (k') can be very small, and have a significant impact on the expected efficiency of macromolecular separations by HPCE.
Though the expectation of 1 to 2 million theoretical plates is unrealistic for HPCE, this does not diminish the impact HPCE may have on the automated separation of macromolecules. Recent publications from biosciences users highlight the advantages of HPCE for solving a specific separation problem which other traditional techniques were not able to solve. Holzman et al., J. Biol. Chem., 266 (1991), pp. 2474-2479; Tran et al., J. Chem., 524 (1991), pp. 459-471; and Lal et al., Arch. Oral. Biol., (1992), pp. 7-13. The major obstacle on the effectiveness of the technique is the reproducibility of the column technology.
Jorgenson et al. had noted that separation of model proteins, such as cytochrome, lysozyme, and ribonuclease A, in untreated fused silica capillaries with a phosphate buffer at pH 7 was accompanied by strong tailing, and suggested this might be caused by Coulombic interaction of the positively charged proteins and the negatively charged capillary wall. Jorgenson et al., Science, 222 (1983), page 266.
Lauer et al., Anal. Chem., 58 (1986), page 166, has reported that the Coulombic repulsion between some idealized proteins and the capillary wall of silica capillaries can overcome adsorption tendencies of these proteins with the capillary wall. They demonstrated separations of model proteins (ranging in molecular weight from 13,000 to 77,000) by varying the solution pH relative to the isoelectric point (pI) of the proteins to change their net charge. However, disadvantages of this approach are that (1) silica begins to dissolve above pH 7, which shortens column life and degrades performance, (2) only proteins with pI's less than the buffer pH can be analyzed, which drastically reduces the range of useful analysis, and (3) interactions which are not Coulombic may still occur even with proteins bearing a net negative charge due to the complexity of protein composition and structure.
Another approach to the problem of biopolymer or protein interactions has been to increase ionic strength. It has been demonstrated that this concept works in principle, but heating is also increased as ionic strength is increased. This heating tends to degrade the efficiency of separation. Increasing salt also decreases the mobility differences among proteins of similar charge and size thus further reducing separation efficiency.
Yet another approach to the problem of undesirable protein interactions with the capillary wall has been to coat the electrophoresis tube with a monomolecular layer of non-crosslinked polymer. Thus, U.S. Pat. No. 4,680,201, inventor Hjerten, issued Jul. 14, 1987, describes a method for preparing a thin-wall, narrow-bore capillary tube for electrophoretic separations by use of a bifunctional compound in which one group reacts specifically with the glass wall and the other with a monomer taking part in a polymerization process. This procedure results in a polymer coating, such as polyacrylamide coating, and is suggested for use in coating other polymers, such as poly(vinyl alcohol) and poly(vinylpyrrolidone). However, this method of capillary tube treatment tends to destroy the electroosmotic flow, and efficiencies are still rather low. These rather low efficiencies suggest that undesirable protein-wall interactions are still occurring.
More recently, in U.S. Pat. No. 4,931,328, issued Jun. 5, 1990, inventor Swedberg disclosed a method for preparing capillary tubes capable of exhibiting reduced protein interactions and controllable electroosmotic flow during electrophoresis. The capillary tube includes an interfacial layer that is covalently bonded to the inner wall of the capillary tube. The interfacial layer has a hydratable amphoteric phase that has a determinable isoelectric point and permits electroosmotic flow control by selection of solution pH. Employing this technique, Maa et al. demonstrated changes in protein selectivity on two deactivated capillaries due to the minimal protein-wall interactions. J. High Resol. Chromatog., 14 (1991), pp. 65-67.
Another recent method of reducing interactions of protein solutes with capillary bore surfaces was disclosed in U.S. Pat. No. 5,006,313, inventor Swedberg, issued Apr. 9, 1991, in which the bore wall of a capillary tube is coated with a reduced interaction phase that includes a plurality of halogen atoms.